Our proposed multidisciplinary investigations have the long-term objective of identifying and validating specific microbiota and metabolomic profiles that can predict loss of tolerance in infants genetically at risk of autoimmunity in order to implement early preventive interventions to re-establish tolerance and ultimately prevent autoimmunity. We will focus our research effort on celiac disease (CD), a unique model of autoimmunity for which the triggering environmental factor (ingestion of gluten-containing grains), a close genetic association with HLA genes (DQ2 or DQ8), and a highly specific humoral autoimmune response (autoantibodies to tissue transglutaminase) are known. Our recent studies have subverted the previous notion that loss of gluten tolerance occurs at the time of its introduction into the child's diet; rather it can occur at any time in life as a consequence of other environmental stimuli. Our preliminary data also suggest that gut microbiome composition and consequent changes in specific metabolomic pathways may contribute to the switch from tolerance to immune response to gluten. To achieve our objective, we will perform a prospective observational study on 500 infants at risk of CD. We will compare the microbiome, metabolome, and immune profiles of infants who develop CD with age- and sex-matched controls (both HLA DQ2/DQ8 negative and positive infants who do not develop the disease) in order to challenge three specific aims. With Aim 1 we propose to study the microbiomic and metatranscriptomic profiles of CD in at-risk infants to define the genetic makeup of these microbiota in association with the development of CD autoimmunity. Changes in microbiomic and metatranscriptomic profiles over time will be analyzed in relation to mode of delivery, exposure to antibiotics, and feeding regimens, including breast feeding and timing of gluten introduction. These studies will be based on the model of interaction between genetic background and environmental pressure in infants at risk of CD. With Aim 2 we will study the infants' metabolomics phenotype variation in relation to tolerance vs. immune response leading to the autoimmune intestinal insult typical of CD. We will establish an in-depth characterization of the infants' metabotypes (microbe-derived metabolomes) and link those data with microbiomic composition and genomic information to build top-down system models of integrated metabolomic phenotypes. These phenotypic models will be interrogated with respect to outcome (tolerance vs. immune response) to obtain predictive models and mechanistic insight into predisposing factors leading to CD autoimmunity. With Aim 3 we will mechanistically link the identified metabolomic products unique to those infants developing CD to intestinal biological events including modulation of intestinal paracellular permeability (Subaim 3a), mucosal regulatory T cell (Treg) functions and mucosal cytokines expression (Subaim 3b), and intestinal stem cell niche gene expression (Subaim 3c).